GABA and L-glutamic acid analogs for antiseizure treatment

ABSTRACT

A compound of the formulawherein R1 is a straight or branched alkyl group having from 1 to 6 carbon atoms, phenyl, or cycloalkyl having from 3 to 6 carbon atoms; R2 is hydrogen or methyl; and R3 is hydrogen, methyl or carboxyl; which is useful in the treatment of seizure disorders. Processes are disclosed for the preparation of the compound. Intermediates prepared during the synthesis of the compound are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/899,918, filed Jul. 24, 1997, now abandoned, which is a divisional ofU.S. patent application Ser. No. 08/420,905, filed Apr. 11, 1995, nowU.S. Pat. No. 6,197,819, which Is a continuation of U.S. patentapplication Ser. No. 08/064,285, filed May 18, 1993, abandoned, which isa continuation-in-part of U.S. patent application Ser. No. 07/886,080,filed May 20, 1992, abandoned, which is a continuation-in-part of U.S.patent application Ser. No. 07/618,692, filed Nov. 27, 1990, abandoned,all of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to novel compounds that are analogs ofglutamic acid and gamma-aminobutyric acid (GABA). More specifically, theanalogs are useful as antiseizure therapy for central nervous systemdisorders such as epilepsy, Huntington's chorea, cerebral ischernia,Parkinson's disease, tardive dyskinesia, and spasticity. It is alsopossible that the present invention could be used as an antidepressant,anxiolytic, and antipsychotic activity.

BACKGROUND OF THE INVENTION

Gamma aminobutyric acid (GABA) and glutamic acid are two majorneurotransmitters involved in the regulation of brain neuronal activity.GABA is the major inhibitory neurotransmitter and L-glutamic acid is anexcitatory transmitter (Roberts E, et al, GABA in Nervous SystemFunction, Raven Press: New York, 1976; McGeer E G, et al, Glutamine,Glutamate, and GABA in the Central Nervous System; Hertz L, Kvamme E,McGeer E G, Schousbal A, eds., Liss: New York, 1983;3-17). An imbalancein the concentration of these neurotransmitters can lead to convulsivestates. Accordingly, it is clinically relevant to be able to controlconvulsive states by controlling the metabolism of thisneurotransmitter. When the concentration of GABA diminishes below athreshold level in the brain, convulsions result (Karlsson A, et al,Biochem. Pharmacol 1974;23:3053-3061). When the GABA levels rise in thebrain during convulsions, seizures terminate (Hayashi T J, Physiol.(London) 1959;145:570-578). The term seizure as used herein meansexcessive unsynchronized neuronal activity that disrupts normal neuronalfunction. In several seizure disorders there is concomitant with reducedbrain GABA levels a diminished level of L-glutamic acid decarboxylase(GAD) activity also observed (McGeer P O, et al, In: GABA in NervousSystem Function; Roberts E, Chase T N, Tower D B, eds., Raven Press: NewYork 1976:487-495; Butterworth J, et al, Neurochem. 1983;41:440-447;Spokes E G, Adv. Exp. Med. Biol. 1978;123:461-473; Wu J Y, et al,Neurochem. Res. 1979;4:575-586; and Iversen L L, et al, Psychiat. Res.1974;11:255-256). Often, the concentrations of GAD and GABA vary inparallel because decreased GAD concentration results in lower GABAproduction.

Because of the importance of GABA as an inhibitory neurotransmitter, andits effect on convulsive states and other motor dysfunctions, a varietyof approaches have been taken to increase the brain GABA concentration.For example, the most obvious approach was to administer GABA. When GABAis injected into the brain of a convulsing animal, the convulsions cease(Purpura D P, et al, Neurochem. 1959;3:238-268). However, if GABA isadministered systematically, there is no anticonvulsant effect becauseGABA, under normal circumstances, cannot cross the blood brain barrier(Meldrum B S, et al, Epilepsy; Harris P, Mawdsley C, eds., ChurchillLivingston: Edinburg 1974:55. In view of this limitation, there arethree alternative approaches that can be taken to raise GABA levels.

The most frequent approach is to design a compound that crosses theblood brain barrier and then inactivates GABA aminotransferase. Theeffect is to block the degradation of GABA and thereby increase itsconcentration. Numerous mechanism-based inactivators of GABAaminotransferase are known (Silverman R B, Mechanism-Based EnzymeInactivation: Chemistry and Enzymology, Vol. I and Il, CRC: Boca Raton1988).

Another approach is to increase GABA concentrations in the brain bymaking GABA lipophilic by conversion to hydrophobic GABA amides (KaplanJ P, et al, G.J. Med. Chem. 1980;23:702-704; Carvajal G. et al, Biochem.Pharmacol. 1964;13:1059-1069; Imines: Kaplan J P, Ibid.; or GABA esters:Shashoua V E, et al, J. Med. Chem. 1984;27:659-664; and PCT PatentApplication WO85/00520, published Feb. 14, 1985) so that GABA can crossthe blood brain barrier. Once inside the brain, these compounds requireamidase and esterases to hydrolyze off the carrier group and releaseGABA.

Yet another approach is to increase brain GABA levels by designing anactivator of GAD. A few compounds have been described as activators ofGAD. The anticonvulsant agent, maleicimid, was reported to increase theactivity of GAD by 11% and as a result increase GABA concentration inthe substantia nigra by up. to 38% (Janssens de Varebeke P, et al,Biochem. Pharmacol. 1983;32:2751-2755. The anticonvulsant drug sodiumvalproate (Loscher W, Biochem. Pharmacol. 1982;31:837-842; Phillips N I,et al, Biochem. Pharmacol, 1982;31:2257-2261) was also reported toactivate GAD and increase GABA levels.

The compounds of the present invention have been found to activate GADin vitro and have a dose dependent protective effect on-seizure in vivo.

Also, the compounds of the present invention have been found to bind anovel binding site which was identified to bind tritiated gabapentin.Gabapentin has been found to be an effective treatment for theprevention of partial seizures in patients refractory to otheranticonvulsant agents. Chadwick D, Gabapentin, pp. 211-222, In: RecentAdvances in Epilepsvy, Vol. 5, Pedley T A, Meldrum B S, (eds.) ChurchillLivingstone, N.Y. (1991). The novel binding site labeled by tritiatedgabapentin was described in membrane fractions from rat brain tissue andin autoradiographic studies in rat brain sections, Hill D, Ibid. Thisbinding site has been used to evaluate the compounds of the presentinvention.

The novel compounds of the present invention are set forth below asFormula I. It should be noted that the compound of Formula I wherein R₁is methyl and each of R₂ and R₃ is hydrogen is taught in Japan PatentNumber 49-40460.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided compounds ofthe Formula I

wherein R₁ is a straight or branched alkyl of from 1 to 6 carbons,phenyl or cycloalkyl having from 3 to 6 carbon atoms; R₂ is hydrogen ormethyl; and R₃ is hydrogen, methyl, or carboxyl; with the proviso thatwhen each of the R₂ and R₃ is hydrogen, R₁ is other than methyl.Pharmaceutically acceptable salts of the compounds of Formula I are alsoincluded within the scope of the present invention. Also included withinthe scope of the present invention are the individual enantiomericisomers of the compounds of the Formula I.

The present invention also provides pharmaceutical compositions of thecompounds of Formula I.

Also provided as a part of the present invention is a novel method oftreating seizure disorders in a patient by administering to said patientan anticonvulsant effective amount of a compound of the followingFormula II

wherein R₁₁ is a straight or branched alkyl of from 1 to 6 carbon atoms,phenyl, or cycloalkyl having from 3 to 6 carbon atoms; R₁₂ is hydrogenor methyl; and R₁₃ is hydrogen, methyl, or carboxyl; or an individualenantiomeric isomer thereof; or a pharmaceutically acceptable saltthereof.

Also, the present invention provides a method for increasing brainneuronal GABA and provides pharmaceutical compositions of the compoundsof Formula II.

The present invention provides novel processes for the synthesis ofchiral Formula I compounds.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a series of3-alkyl-4-aminobutyric acid or 3-alkyl glutamic acid analogs which areuseful as anticonvulsants. Illustrative of the alkyl moieties asrepresented by R₁ and R₁₁ in Formulas I and II are methyl, ethyl,propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, isopentyl,and neopentyl as well as other alkyl groups. The cycloalkyl groupsrepresented by R₁ and R₁₁ in Formulas I and II are exemplified bycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The analogs arefurther shown herein to prevent seizure while not causing the sideeffect of ataxia, such a side effect being found in several anti-seizurepharmaceuticals.

The more preferred compounds of the present invention are of Formula Iabove wherein R₃ is hydrogen, R₂ is hydrogen, and R₁ isobutyl.

That is, the preferred compound is 4-amino-3-(2-methylpropyl)butanoicacid. It has been found that this compound is unexpectedly more potentthan the other analogs synthesized in accordance herewith and tested invivo. What is further surprising, as the following data shows, is thatthis preferred compound is the least effective one of the analogs testedin activating GAD in vitro. Accordingly, it was very unexpected thatthis preferred compound had such a high potency when tested in vivo.

The most preferred compounds of the present invention are the (S)-(+)-and the (R)(−)-4-amino-3-(2-methylpropyl)butanoic acid with the(S)-(+)-enantiomer being most preferred. The (S)-(+)-enantiomer wasfound to be the most potent compound within the scope of the presentinvention for displacement of tritiated gabapentin, and both the(S)-(+)- and the (R)-(−)-enantiomers showed pronounced stereoselectivityfor both displacement of tritiated gabapentin and for anticonvulsantactivity in vivo.

The compounds made in accordance with the present invention may formpharmaceutically acceptable salts with both organic and inorganic acidsor bases. For example, the acid addition salts of the basic compoundsare prepared either by dissolving the free base in aqueous or aqueousalcohol solution or other suitable solvents containing the appropriateacid and isolating the salt by evaporating the solution. Examples ofpharmaceutically acceptable salts are hydrochlorides, hydrobromide,hydrosulfates, etc, as well as sodium, potassium, and magnesium, etc,salts.

The method for the formation of the 3-alkyl-4-aminobutanoic acidsstarting from 2-alkanoic esters is prepared from commercially availablealdehydes and monomethyl malonate by the Knoevenagel reaction, (Kim Y C,et al, J. Med. Chem. 1965:8509) with the exception of ethyl4,4-dimethyl-2-pentenoate.

More specifically, the following is a procedure which can be generallyapplied to the preparation of all the 3-alkylglutamic acids. Ten gramsof a 3-alkyl-5,5-dicarbethoxy-2-pyrrolidinone was refluxed in 150 mL of49% fuming HBr for 4 hours. After this time, the contents were placed inan evaporator and the volatile constituents were removed in vacuo withthe aid of a hot-water bath. The gummy residue was dissolved in 25 mL ofdistilled water and the water was removed with the aid of theevaporator. This process was repeated once more. The residue wasdissolved in 20 mL of water, and the pH of the solution was adjusted to3.2 with concentrated NH₃ solution. At this point the chain length ofthe individual 3-alkylglutamic acids altered the solubility so thatthose whose side chains were larger precipitated with the ease fromsolution. Precipitation of the alkylglutamic acids with smallersubstituents (methyl, ethyl, and propyl) could be encouraged by coolingon an ice bath or by diluting the aqueous solution with 100 mL ofabsolute ethanol. Precipitation from the water-alcohol mixture iscomplete in 48 hours. Care must be taken to add the ethanol slowly toprevent the precipitation of an amorphous solid which is notcharacteristic of the desired 3-alkylglutamic acids. Samples of theamino acids were purified for analysis by recrystallizing from awater-ethanol mixture. All melted with decomposition. Melting points ofthe decomposed 3-alkylglutamic acids corresponded with those of theirpyroglutamic acids.

Ethyl 4,4-dimethyl-2-pentenoate was prepared from 2,2-dimethylpropanoland ethyl lithioacetate, followed by dehydration of the β-hydroxy esterwith phosphoryl chloride and pyridine.

The Michael addition of nitromethane to alpha, β-unsaturated compoundsmediated by 1,1,3,3-tetramethylguanidine or1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU) afforded 4-nitroesters in goodyields. More specifically, a mixture of nitromethane (5 mol),α,β-unsaturated ester (1 mol), and tetramethyl-guanidine (0.2 mol) wasstirred at room temperature for 2 to 4 days. (In case of methylacrylate, the ester has to be added at a temperature below 300.) Theprogress of the reaction was followed by IR (disappearance of the C═Cband) and G.L.C. analysis. The reaction mixture was washed with dilutehydrochloric acid and extracted with ether. The organic extract wasdried, the solvent removed at reduced pressure, and the 20 residuedistilled at a pressure of 2 torr. Although the aliphatic nitrocompounds are usually reduced by either high pressure catalytichydrogenation by metal-catalyzed transfer hydrogenation, or by newlyintroduced hydrogenolysis methods with ammonium formate or sodiumborohydride and palladium as catalysts, applicants have found that4-nitrocarboxylic esters can be reduced almost quantitatively to thecorresponding 4-aminocarboxylic esters by hydrogenation using 10%palladium on carbon as catalysts in acetic acid at room temperature andatmospheric pressure. The amino esters produced were subjected to acidhydrolysis to afford the subject inventive compounds in good yields.This procedure provides access to a variety of 3-alkyl-4-aminobutanoicacids as listed in Tables 1 and 2 as examples and thus is advantageousin comparison to methods previously used.

Examples of more specific methods of making compounds in accordance withthe present invention are as follows, optionally utilizing the methodsdescribed in detail above. When the starting material is notcommercially available, the synthetic sequence may be initiated with thecorresponding alcohol, which is oxidized to the aldehyde by the methodof Corey E J, et al, Tetrahedron Lett, 1975:2647-2650.

The chiral compounds of Formulas I and II are prepared as set forth inthe schematic in Chart I hereof. Although the schematic in Chart Idepicts the chiral synthesis of specific compound(S)-(+)-4-amino-3-(2-methylpropyl)butanoic acid, one skilled in the artcan readily see that the method of synthesis can be applied to anydiastereomeric compound of Formulas I and II.

In Chart I Ph is phenyl, Bn is benzyl, THF is tetrahydrofuran, LDA islithium diisopropylamide, BH₃.SMe₂ is borane dimethyl sulfide complex,TsCl is tosyl chloride, and DMSO is dimethylsulfoxide.

The detailed synthetic procedure is set forth hereinbelow in Example 1.The key introductory literature for this methodology was discussed inEvans' paper, J. Am. Chem. Soc. 1982;104:1737-9. The metal enolate canbe formed with a lithium or sodium amide base, and subsequentlyalkylated to give an a substituted carboxylic acid derivative. Thismethodology was valuable for the enantioselective synthesis of theseα-substituted carboxylic acid derivatives. In this seminal paper, Evansdescribed the preparation of propionic acid derivatives with a series ofsimple alkylating agents. By varying the stereochemistry of the chiralsynthon (the oxazolidinone), he was able to get high stereoselectivity.

Evans has used this chiral auxiliary in other synthetic studies, butnone has been related to 4-amino-3-(2-methylpropyl)butanoic acid whichcontains a β-substituted-γ-amino acid. The methodology as presented byEvans teaches toward α-substitution, and away from β-substitution, andhas not been used in the preparation of this type of unusual amino acid.N-acyloxazolidinones have been used to form chlorotitanium enolates thathave been reacted with Michael adducts such as acrylonitrile, J. Org.Chem. 1991;56:5750-2. They have been used in the synthesis of therutamycin family of antibiotics, J. Org. Chem. 1990;55:6260-8 and instereoselective aldol condensations, Org. Synth. 1990;68:83-91. Chiralα-amino acids were prepared via the oxazolidinone approach. In thissequence, a dibutylboron enolate was brominated and displaced withazide, Tetrahedron Lett. 1987;28:1123-6. Other syntheses ofβ-hydroxy-α-amino acids were also reported via this chiral auxiliarythrough aldol condensation (Tetrahedron Lett. 1987;28:39-42; J. Am.Chem. Soc. 1987;10:7151-7). α,β-Unsaturated N-acyloxazolidinones havealso been used to induce chirality in the Diels-Alder reaction, (J. Am.Chem. Soc. 1988;110:1238-56. In none of these examples, or others foundin the literature, is this methodology used to prepare (β-substitutedcarboxylic acids or 3-substituted GABA analogs.

In another embodiment, the chiral compounds of Formulas I and II can beprepared in a manner which is similar to the synthesis depicted in ChartI. In this embodiment, however, step 8 in Chart I is replaced by analternate two step procedure which is set forth hereinbelow in Example 2(sodium hydroxide is preferred, however, other solvents known to thoseof skill in the art which can hydrolyze the azide (8) to intermediateazide (8a) can be employed). Instead of reducing the azide (8) to theamino acid (9) in Chart I, the alternate procedure hydrolyzes the azide(8) to give an intermediate azide (8a) which is subsequently reduced(see Chart Ia).

There are two major advantages to hydrolyzing azide (8) to give theintermediate azide (8a) prior to reduction. The first advantage is thatintermediate azide (8a) may be purified by extraction into aqueous base.After the aqueous extract is acidified, intermediate azide (8a) may beextracted into the organic phase and isolated. This allows for apurification of intermediate azide (8a) which does not involvechromatography. The purification of azide (8) requires chromatographywhich is very expensive and often impractical on a large scale.

The second advantage is that intermediate azide (8a) may be reduced toamino acid (9) without added acid. Reduction of azide (8) requiresaddition of acid, e.g., hydrochloric acid in order to obtain amino acid(9). Unfortunately, lactamization of amino acid (9) is promoted by thepresence of acid. Intermediate azide (8a) may be reduced under nearneutral conditions to give amino acid (9), thus minimizing the problemof lactam formation.

In another preferred embodiment, the chiral compounds of Formulas I andII can be prepared as set forth in the Schematic in Chart II hereof.Although the schematic in Chart II depicts the chiral synthesis ofspecific compound (S)-(+)-4-amino-3-(2-methylpropyl)butanoic acid, oneskilled in the art can readily see that the method of synthesis can beapplied to any diastereomeric compound of Formulas I and II.

In Chart II Ph is phenyl, and Ts is tosyl.

The detailed synthetic procedure is set forth hereinbelow in Example 3.This procedure is similar to the synthesis route depicted in Chart I,however, the procedure of Chart II replaces the benzyl ester in thesynthesis route of Chart I with a t-butyl ester. The desired amino acid(9) and (109) is the same end product in both Charts I and II,respectively.

There are several advantages to using the t-butyl ester rather than thebenzyl ester in the synthesis of amino acid (9) or (109). A firstadvantage relates to the hydrolysis of the chiral auxiliary in step 4 ofChart 1. During the hydrolysis of the chiral auxiliary in this reactionsome hydrolysis of the benzyl ester often occurs. Hydrolysis of thet-butyl ester in Chart II has not been experienced.

Another advantage relates to the use of alcohol (106) in Chart II overthe use of alcohol (6) in Chart I. A problem with the benzylester-alcohol is the tendency of the benzyl ester-alcohol to undergolactonization as shown below. Although lactonization of the benzyl estercan be avoided under some conditions, the t-butyl ester-alcohol is farless prone to lactonization.

Still another advantage, which was previously discussed with regard tothe synthetic procedure depicted by Chart Ia, is that the t-butylsynthetic route minimizes the problem of lactam formation of the aminoacid end product (109). Instead of reducing azide (108) to amino acid(109) which requires the addition of acid that causes lactamization ofamino acid (109), azide (108) is first hydrolyzed to intermediate azide(108a). Intermediate azide (108a) may be reduced under neutralconditions to give amino acid (109), thus minimizing the problem oflactam formation.

It should also be mentioned that several novel intermediates areproduced by the processes discussed herein. Some of these intermediateswhich are depicted in Charts I, Ia, and II include in the racemate or Ror S enantiomer form:

4-methyl-5-phenyl-2-oxazolidinone,

4-methyl-(2-methylpropyl)-2-dioxo-5-phenyl-3-oxazolidine butanoic acid,phenylmethyl ester,

4-methyl-pentanoyl chloride,

4-methyl-3-(4-methyl-1-oxopentyl)-5-phenyl-2-oxazolidinone,

2-(2-methylpropyl)-butanedioic acid, 4-(phenylmethyl) ester,

3-(azidomethyl)-5-methyl-hexanoic acid, phenylmethyl ester,

3-(hydroxymethyl)-5-methyl-hexanoic acid, phenylmethyl ester,

5-methyl-3-[[[(4-methylphenyl)sulfonyl]oxy]-methyl]-hexanoic acid,phenylmethyl ester,

3-(azidomethyl)-5-methyl-hexanoic acid,

2-(2-methylpropyl)-1,4-butanedioic acid, 4-(1,1-dimethylethyl) ester,

3-(azidomethyl)-5-methyl-, 1,1-dimethylethyl ester,

3-(hydroxymethyl)-5-methyl-hexanoic acid, 1,1-dimethyl ester,

5-methyl-3-[[[(4-methyl(phenyl)sulfonyl]oxy]-methyl-hexanoic acid,1,1-dimethylethyl ester, or

4-methyl-(2-methylpropyl)-2-dioxo-5-phenyl-3-oxazolidinebutanoic acid,1,1-dimethylethyl ester.

The compounds made by the aforementioned synthetic methods can be usedas pharmaceutical compositions as an antidepressant, anxiolytic,antipsychotic, antiseizure, antidyskinesic, or antisymptomatic forHuntington's or Parkinson's diseases when an effective amount of acompound of the aforementioned formula together with a pharmaceuticallyacceptable carrier is used. That is, the present invention provides apharmaceutical composition for the suppression of seizures resultingfrom epilepsy, the treatment of cerebral ischemia, Parkinson's disease,Huntington's disease and spasticity and also possibly forantidepressant, anxiolytic, and antipsychotic effects. These latter usesare expected due to functional similarities to other known compoundshaving these pharmacological activities. The pharmaceutical can be usedin a method for treating such disorders in mammals, including human,suffering therefrom by administering to such mammals an effective amountof the compound as described in Formulas I and II above in unit dosageform.

The pharmaceutical compound made in accordance with the presentinvention can be prepared and administered in a wide variety of dosageforms. For example, these pharmaceutical compositions can be made ininert, pharmaceutically acceptable carriers which are either solid orliquid. Solid form preparation include powders, tablets, dispersiblegranules, capsules, cachets, and suppositories. Other solid and liquidform preparations could be made in accordance with known methods of theart. The quantity of active compound in a unit dose of preparation maybe varied or adjusted from 1 mg to about 300 mg/kg (milligram perkilogram) daily, based on an average 70 kg patient. A daily dose rangeof about 1 mg to about 50 mg/kg is preferred. The dosages, however, maybe varied depending upon the requirement with a patient, the severity ofthe condition being treated, and the compound being employed.Determination of the proper dosage for particular situations is withinthe skill of the art.

Illustrative examples of compounds made in accordance with the presentinvention were tested to demonstrate the ability of the compounds toactivate GAD in vitro and to prevent seizure in vivo without the sideeffect of ataxia.

In Vitro GAD Activation

Assays were carried out in 10 mL vials sealed with serum caps throughwhich a center well (Kontes Catalog No. 882320-000) was inserted. Thecenter well was charged with 200 μL of freshly prepared 8% KOH solution.Various concentrations of L-glutamic acid (0.5, 0.25, 0.166, 0.125, 0.10mM) containing [¹⁴C]L-glutamate (10 μCi/mmol) in 50 mM potassiumphosphate buffer, pH 7.2 were shaken at 37° C. in separate vials withpurified L-glutamic acid decarboxylase (18.75 μg; spec. act 10.85μmol/min mg) in a total volume of 2.00 mL. After being shaken for 60minutes, the enzyme reactions were quenched by the addition of 200 μL of6 M sulfuric acid to the contents of each of the vials. The vials wereshaken for an additional 60 minutes at 37° C. The center wells wereremoved and placed in scintillation vials with 10 mL of scintillationfluid for radioactivity determination. The same assays were repeatedexcept in the presence of various concentrations of the activators (2.5,1.0, 0.5, 0.25, 0.1, 0.05 mM). The Vmax values were determined fromplots of 1/cpm versus 1/[glutamate] at various concentrations ofactivators. The data were expressed as the ratio of the Vmax in thepresence of the activators to the Vmax in the absence of the activatorstimes 100%.

The results of the experiment are shown in Table 1. The tests show thatthere was significant activation by the various compounds tested todiffering degrees. The known activator sodium valproate and gabapentinwere tested.

In vivo tests were performed to demonstrate the seizure preventingcapabilities of the novel compounds. Threshold maximum electroshock isan animal model test for generalized seizures that is similar to that ofPiredda S G, et al, Pharmacol. and Exptl. Therap. 1985;232(3):741-45.The methods for this test are described as follows.

Male CF-1 mice (22-30 g) were allowed free access to food and waterprior to testing. For screening, groups of five mice were given acompound intravenously at doses of 30, 100, and 300 mg/kg and tested at0.5, 2.0, and 4.0 hours after dosing. Drugs were either dissolved in0.9% saline or suspended in 0.2% methylcellulose. Animals were shockedwith corneal electrodes (see below) and observed for tonic hindlimbextensor seizures. Absence of hindlimb extension was taken as ananticonvulsant effect.

The electroshock apparatus delivered a 60 Hz sine wave with a currentamplitude of 14 mA (peak-to-peak) for 0.2 seconds. The current strengthof 14 mA used in this procedure produced tonic extensor seizures inapproximately 95% of untreated mice, but was only slightly abovethreshold for tonic extension.

Summaries of the numbers of animals protected from seizures when tested120 minutes after administration of each compound set forth in theleft-hand column are given in Table 2 for varying dose levels set forthin the second column of the table.

Due to the interesting phenomena related to the (R,S)-i-butyl GABA (thecompound having significantly higher potency and effectiveness withoutcausing ataxia), threshold maximal electroshock tests where conductedvarying the time of testing from 1 hour to 8 hours, the dose being 10mg/kg in mice, injected intravenously. Table 3 shows the results ofthese tests indicating a maximum protection after 2 hours of testing.

In view of the above results, a dose response curve was made for the twohour testing time period in mice, the drug being given intravenously at10 mg/kg. The results of this test is shown in Table 4 with a calculatedEDSO equaling 2.07 mg/kg.

A third pharmacological test was performed as described in Krall R L, etal, Epilensia. 1978;19:409. In this procedure, drugs were tested forattenuation of threshold clonic seizures in mice caused by subcutaneousadministration of pentylenetetrazol (85 mg/kg) which is a generallyaccepted model for absence type seizures. Results from the third testfor the compound when administered either intravenously or orally isshown in Table 5. The test was conducted at three dose levels, showingeffective protection at 30 mg/kg and 100 mg/kg with no ataxia.

The above is a significant finding because the compound having the leastability to activate GAD in vitro surprisingly had an approximately10-fold increase in potency over the other compounds tested. Even moreunexpected is the absence of ataxic side effect coupled to this increasein potency.

TABLE 1 Activation of GAD by GABA analogs at various concentrationsexpressed in %

2.5 1.0 0.5 0.25 0.1 0.05 R₁, R₂ mM mM mM mM mM mM (R,S)-CH₃, H 239 168142 128 118 107 (R)-CH₃H 327 202 185 135 128 109 (S)-CH₃H 170 118 — 103— — CH₃, CH₃ 174 125 — 109 — — (R,S)-C₂H₅, H 172 128 — 108 — —(R,S)-n-C₃H₇, H 156 112 — 105 — — (R,S)-i-C₃H₇, H 140 108 — 104 — —(R,S)-n-C₄H₉, H 178 117 — 105 — — (R,S)-i-C₄H₉, H 143 113 — 109 — —(R,S)-s-C₄H₉, H 169 119 — 105 — — (R,S)-t-C₄H₉, H 295 174 147 121 117108 (R,S)-neo-C₅H₁₁, H 279 181 — 130 — — (R,S)-i-C₅H₁₁, H 142 118 — 109— — (R,S)-C₆H₁₁, H 125 100 — 100 — — (R,S)-C₆H₅, H 218 129 — 110 — —

2.5 1.0 0.5 0.25 0.1 0.05 R mM mM mM mM mM mM H(R,S) 140 111 — 104 — —H(R) 173 125 — 108 — — H(S) 100 100 — 100 — — CH₃ 143 121 — 109 — — C₆H₅207 151 — 112 — — Sodium Valproate 207 138 124 119 115 105 GABAPENTIN178 145 — 105 — — Activation of GAD by glutamate analogs expressed in %

R 2.5 mM 1.0 mM 0.25 mM CH₃ 212 144 113 C₂H₅ 170 128 113 n-C₃H₇ 153 125108 i-C₃H₇ 144 114 105 n-C₄H₉ 133 117 105 i-C₄H₉ 129 112 106 C₆H₅ 172135 112 Sodium Valproate 207 138 119

TABLE 2 Prevention of tonic extensor seizures in mice followingintravenous administration of 3-substituted GABA derivatives EffectAtaxia Dose Time After # Protected/ # Ataxia/ R (mg/kg) Dose (min) #Tested # Tested (R,S)-CH₃ 10 120 0/5  0/5  30 120 4/5  0/5  100 120 3/5 0/5  CH₃ 1 120 1/10 0/10 3 120 2/10 0/10 10 120 4/10 0/10 30 120 3/100/10 100 120    3/10 (5/10) 1/10 CH₃ 10 120 1/10 1/10 30 120 2/10 0/10100 120 5/10 0/10 t-C₄H₉ 10 120 2/10 0/10 30 120 2/10 0/10 100 120 5/100/10 C₂H₅ 3 120 1/5  0/5  10 120 1/5  0/5  30 120 2/5  0/5  100 120 5/5 0/5  (CH₃)₂ 30 120 4/5  0/5  100 120 4/5  0/5  n-C₄H₉ 10 120 1/10 0/1030 120 3/10 0/10 100 120 4/10 0/10 s-C₄H₉ 3 120 2/10 0/10 10 120 3/100/10 30 120 2/10 0/10 i-C₄H₉ 0.3 120 1/10 0/10 0.8 120 3/10 0/10 2.0 1205/10 0/10 5.5 120 7/10 0/10 14.4 120 9/10 0/10 n-C₃H₇ 3 120 2/10 0/10 10120 2/10 3/10 100 120 3/10 0/10 i-C₃H₇ 10 120 5/10 1/10 30 120 5/10 0/10100 120 6/10 0/10 C₆H₅ 100 120 0/10 0/10 neo-C₅H₁₁ 10 120 2/10 0/10 30120 4/10 0/10 100 120 4/10 0/10 High-intensity corneal electroshockconsisted of 50 mA, base-to-peak sinusoidal current for 0.2 seconds. Allother data was from low-intensity electroshock, 17 mA base-to-peaksinusoidal current for 0.2 seconds.

TABLE 3 Threshold maximal electroshock with isobutyl GABA Time ofTesting # Protected 1 hr. 2/10 2 hr. 8/10 4 hr. 4/10 8 hr. 2/10

TABLE 4 Threshold maximal electroshock with isobutyl GABA Dose m/k #Protected 0.3 1/10 0.8 3/10 2.0 5/10 5.5 7/10 14.4 9/10

TABLE 5 Maximal electroshock data Effect Ataxia Dose Time After #Protected/ # Ataxia/ R (mg/kg) Dose (min) # Tested # Tested i-C₄H₉ 10120 1/5 0/5 i-C₄H₉ 30 120 4/5 0/5 i-C₄H₉ 100 120 4/5 0/5

As noted hereinabove, the S-(+)enantiomer of4-amino-3-(2-methylpropyl)butanoic acid (3-isobutyl GABA or IBG) whichis structurally related to the known anticonvulsant, gabapentin,potently displaces tritiated gabapentin from a novel high-affinity sitein rat brain membrane fractions. Also the S-(+)enantiomer of 3-isobutylGABA is responsible for virtually all blockade of maximal electroshockseizures in mice and rats. The R(−) enantiomer of 3-isobutyl GABA ismuch less effective in the blockade of maximal electroshock seizures andin displacement of tritiated gabapentin from the novel high-affinitybinding site. Table 6 below sets forth data comparing gabapentin,racemic 3-isobutyl GABA ((±)-IBG), S-(+)-3-isobutyl GABA ((S)-IBG) andR-(−)-3-isobutyl GABA ((R)-IBG) in these assays.

TABLE 6 3-Isobutyl GABA (ED₅₀) Test System Gabapentin (±)-IBG (S)-IBG(R)-IBG Gabapentin Receptor 0.14 μM 0.10 μM 0.044 μM 0.86 μM Binding(IC₅₀) IV Mouse Low-Intensity 4.3 mg/Kg 4.8 mg/Kg 4.0 mg/Kg >100 mg/KgElectroshock IV Mouse Maximal 75 mg/Kg 10 mg/Kg 18 mg/Kg >100 mg/KgElectroshock PO Mouse Maximal 200 mg/Kg 47 mg/Kg 12 mg/Kg Elctroshock IVMouse Ataxia >100 mg/Kg >100 mg/Kg >300 mg/Kg >100 mg/Kg (IP) Timecourse of anticonvulsant activity (all compounds) peaks 2.0 hours afterdose and mostly gone 8 hours after dose.

The data set forth in Table 6 was obtained as follows. Foranticonvulsant testing, male CF-1 strain mice (20-25 g) and maleSprague-Dawley rats (75-115 g) were obtained from Charles RiverLaboratories and were maintained with free access to food and waterbefore testing. Maximal electroshock was delivered with cornealelectrodes by conventional methods (Krall, supra, 1975) except thatlow-intensity electroshock with mice consisted of 17 mA of currentrather than the conventional 50 mA (zero to peak). Briefly, mice weregiven test substance and were tested for prevention of seizures byapplication of electrical current to the corneas by 2 metal electrodescovered with gauze and saturated with 0.9% sodium chloride. Electroshockstimulation was delivered by a constant-current device that produced 60Hz sinusoidal electrical current for 0.2 seconds. For rats, maximalelectroshock stimulation consisted of 120 mA of current. Ataxia in micewas assessed by the inverted screen procedure in which mice wereindividually placed on a 4.0-inch square of wire mesh that wassubsequently inverted (Coughenour, supra, 1978). Any mouse that fellfrom the wire mesh during a 60 second test period was rated as ataxic.ED₅₀ values were determined by probit analysis of results with at least5 dose groups of 10 mice or 8 rats each.

All drugs were freely soluble in aqueous media. For in vivo studies,drug solutions were made in 0.9% sodium chloride and given in a volumeof 1 mL/100 g body weight. Intravenous administration was given by bolusinjection into the retro-orbital sinus in mice. Oral administrationswere by intragastric gavage.

For binding studies, partially purified synaptic plasma membranes wereprepared from rat neocortex using sucrose density gradients. Thecerebral cortex of 10 rats was dissected from the rest of the brain andhomogenized in 10 volumes (weight/volume) of ice-cold 0.32 M sucrose in5 mM tris-acetate (pH 7.4) using a glass homogenizer fitted with ateflon pestle (10-15 strokes at 200 rpm). The homogenate was centrifugedat 100 g for 10 minutes and the supernatant collected and kept on ice.The pellet (PI) was rehomogenized in 20 mL of tris-sucrose and thehomogenate recentrifuged. The combined supernatants were centrifuged at21,500 g for 20 minutes. The pellet (P2) was resuspended in 1.2 Mtris-sucrose and 15 mL of this mixture was added to ultracentrifugetubes. On to this, 10 mL of 0.9 M sucrose was layered followed by afinal layer of 5 mM tris-acetate, pH 8.0. Tubes were centrifuged at100,000 g for 90 minutes. The synaptic plasma membranes located at the0.9/1.2 M sucrose interface were collected, resuspended in 50 mL of 5 mMtris-acetate, pH 7.4, and centrifuged at 48,000 g. The final pellet wasresuspended in 50 mL of tris-acetate, pH 7.4, aliquoted, and then frozenuntil use.

The assay tissue (0.1 to 0.3 mg protein) was incubated with 20 mM[³H]-gabapentin in 10 mM HEPES buffer (pH 7.4 at 20° C., sodium free) inthe presence of varying concentrations of test compound for 30 minutesat room temperature, before filtering onto GFB filters under vacuum.Filters were washed 3 times with 5 mL of ice cold 100 mM NaCl solutionand dpm bound to filters was determined using liquid scintillationcounting. Nonspecific binding was defined by that observed in thepresence of 100 mM gabapentin.

In view of the above demonstrated activity of the compoundscharacterizing the present invention and in particular the4-amino-3-(2-methylpropyl)butanoic acid (isobutyl GABA) the compoundsmade in accordance with the present invention are of value aspharmacological agents, particularly for the treatment of seizures inmammals, including humans.

EXAMPLE 1 (S)-(+)-4-amino-3-(2-methylpropyl)butanoic acid

The following “steps”, refer to Chart I.

Step 1

To a solution of 4-methylvaleric acid (50.0 g, 0.43 mol) in 100 mL ofanhydrous chloroform was added thionyl chloride (60 mL, 0.82 mol). Thereaction mixture was refluxed for 2 hours and then cooled to roomtemperature. Excess chloroform and thionyl chloride was removed bydistillation. The residue oil was then fractionally distilled to give45.3 g (78%) of the acid chloride (2), bp=143-144° C.

Acid chloride (2) was also be prepared by an alternative method whicheliminated use of chloroform which has waste disposal and operatorexposure difficulties. The alternate method also minimized the formationof 4-methylvaleric anhydride.

To a solution of thionyl chloride (98.5 kg, 828 mol) andN,N-dimethylformamide (2 kg, 27 mol) was added 4-methylvaleric acid (74kg, 637 mol) while maintaining a reaction temperature of 25-30° C.Hexanes (30 L) were added and the solution was maintained at 30-35° C.for 1 hour and 15 minutes. The solution was then heated to 70-75° C. for1 hour and 10 minutes. The solution was subjected to atmosphericdistillation until a solution temperature of 95° C. was reached. Aftercooling, hexanes (30 L) were added and the solution was subjected toatmospheric distillation until a solution temperature of 97° C. wasreached. Distillation of the residual oil produced 79 kg (92%) of acidchloride (2), bp=−77° C., 60-65 mm Hg.

Step 2

To a solution of (4R,5S)-(+)-4-methyl-5-phenyl-2-oxazolidinone (5.27 g,29.74 mmol) in 70 mL of anhydrous tetrahydrofuran at −78° C. under argonatmosphere was added a 1.6 M solution of n-butyllithium (19 mL, 30.40mmol) in hexanes slowly. The mixture was allowed to stir at −78° C. for15 minutes then the acid chloride (4.5 g, 33.43 mmol) was added toquench the reaction. The reaction was stirred at −78° C. for 10 minutesthen 0° C. for 30 minutes. A saturated solution of sodium bicarbonate(50 mL) was added and the mixture was stirred at 0° C. for 30 minutes.The organic layer was collected and the aqueous layer was extracted withethyl acetate (3×). The organic extracts were combined and dried withanhydrous magnesium sulfate. It was then filtered and concentrated togive a colorless oil. The oil was then chromatographed with 8% ethylacetate in hexanes on silica gel to give 7.56 g (82%) of theacyloxazolidinone (3) as a white solid. Anal. Calcd for C₁₆H₂₁NO₃: C,69.79; H, 7.69; N, 5.09. Found: C, 69.56; H, 7.63; N, 5.06.

Acyloxazolidinone (3) was also prepared by an alternate method which wasconducted at −5° C. to 0° C. rather than −78° C. which is difficult andexpensive to achieve on a manufacturing scale. The alternate method alsogave a crystalline solid from the reaction mixture rather than an oilwhich must be chromatographed.

To a solution of 4-methyl-5-phenyl-2-oxazolidinone (64 g, 0.36 mol) inanhydrous tetrahydrofuran (270 g) at −5° C. was added a 15% solution ofn-butyllithium in hexane (160 g, 0.37 mol) over a temperature range of−5° C. to 0° C. Acid chloride (2) (48.6 g, 0.36 mol) was added at −10°C. to 0° C. The reaction was quenched with a solution of water (90 mL)and sodium bicarbonate (4 g). Ethyl acetate (200 g) was added and thelayers were separated. The organic layer was extracted with water (2×50mL) and the aqueous phases were back extracted with ethyl acetate (100g). The organic extracts were combined and approximately 150 mL ofsolvent was removed by distillation. Atmospheric distillation wascontinued and heptane (2×200 g) was added until a vapor temperature of95° C. was reached. The solution was cooled to 5° C. The product wascollected by filtration, washed with cold heptane, and dried to give 79g (80%) of acyloxazolidinone (3).

Step 3

To a solution of diisopropylamine (4.8 mL, 34.25 mmol) in 30 mL ofanhydrous tetrahydrofuran at 0° C. under argon atmosphere was added a1.6 M solution of n-butyllithium (21 mL, 33.60 mmol) in hexanes slowly.The solution was stirred at 0° C. for 30 minutes then cooled to −78° C.A solution of the acyloxazolidinone (3) (7.56 g, 27.46 mmol) in 30 mL ofanhydrous tetrahydrofuran was added and the pale yellow solution wasstirred at −78° C. for 30 minutes. Benzyl α-bromoacetate was added andthe resulting solution was stirred at −25° C. for 2 hours. The reactionmixture was quenched with a half-saturated ammonium chloride solutionand extracted by ethyl acetate (2×). The combined organic layers weredried with anhydrous magnesium sulfate and then filtered andconcentrated to give a colorless oil. The oil was then chromatographedwith 8% ethyl acetate in hexanes on silica gel to give 6.16 g (53%) ofthe acyloxazolidinone (4) as a white solid. Anal. Calcd for C₂₅H₂₉NO₅:C, 70.90; H, 6.90; N, 3.31. Found: C, 70.47; H, 6.87; N, 3.45.

Acyloxazolidinone (4) was also prepared by an alternate method that wasadvantageous in that the reaction was conducted a higher temperature(−35° C. to −25° C. rather than −78° C.) and an expensive and difficultchromatographic separation was avoided.

Acyloxazolidinone (3) (85 kg, 308 mol) was dissolved in anhydroustetrahydrofuan (201 kg) and cooled to −30° C. Lithium diisopropyl amine(340 mol) in methyl-t-butyl ether/hexane was added while maintaining areaction temperature of −35° C. to −25° C. Benzyl bromoacetate (85 kg,371 mol) was then added while maintaining a reaction temperature of −35°C. to −25° C. Water (60 kg) and methyl-t-butyl ether (93 kg) were addedand the mixture was allowed to warm to 18° C. The layers were separatedand the organic layer was extracted with a solution of water (40 L) andsodium chloride (7 kg). The layers were separated and the organic layerwas concentrated to 200 liters by distillation. Isopropyl alcohol (200L) was added and the solution was again concentrated to 200 liters bydistillation. Isopropyl alcohol (425 L) and water (160 L) were added andthe mixture was heated to 50° C. The solution was cooled to 18° C. Theproduct was collected by filtration, washed with isopropyl alcohol/waterand dried under reduced pressure to give 58.7 kg (49% yield) ofacyloxazolidinone (4) as a solid.

Step 4

To a pre-cooled (0° C.) solution of the acyloxazolidinone (4) (24.3 g,57.38 mmol) in 600 mL of tetrahydrofuran was added a solution of 30%hydrogen peroxide (23.7 mL) in 320 mL of 0.2 M lithium hydroxidesolution via a dropping funnel in 20 minutes. The reaction mixture wasallowed to stir at 0° C. for 4 hours. A solution of sodiummeta-bisulphite (62.2 g, 0.33 mol) in 320 mL of water was then addedslowly to quench the reaction. The mixture was stirred at 0° C. for 20minutes. Excess tetrahydrofuran was removed on a rotavap. The aqueousresidue was extracted with ethyl acetate (3×350 mL). The combinedorganic extracts were dried with anhydrous magnesium sulfate and thenfiltered. The oily residue after concentration was chromatographed by40% ethyl acetate in hexane on silica gel to give 13.34 g (88%) of theacid (5) as a clear oil. The column was then eluted with 50% ethylacetate in hexane to give the oxazolidinone chiral auxiliary.

¹H NMR (300 MHz, CDCl₃) of acid (5): δ9.80 (br s, 1H), 7.36 (m, 5H),5.14 (narrow ABq, 2H, J_(AB)=11.4 Hz), 2.80 (m, 1H), 2.63 (ABX, 2H,J_(AB)=16.75 Hz, J_(AB)=9.13 Hz, J_(BX)=5.16 Hz, U_(AB)=73.20 Hz) , 1.66(m, 2H), 1.33 (m, 1H), 0.93 (d, 3H, J=7.32 Hz), 0.91 (d, 3H, J=6.45 Hz).

In an alternate method, after concentration of the reaction to an oilyresidue, hexane or heptane may be added in order to precipitate theoxazolidinone chiral auxiliary. Filtration then yields the chiralauxiliary in 80% recovery. The hexane or heptane filtrate containingacid (5) is then extracted with either an ethanol water solution or withwarm water to remove any remaining chiral auxiliary. This alternativemethod avoids a costly and difficult chromatographic separation of thechiral auxiliary from the acid (5).

Step 5

To a solution of the acid (5) (13.34 g, 50.47 mmol) in 460 mL anhydroustetrahydrofuran at 0° C. under argon was added borane dimethyl sulfidecomplex (10 M, 11.2 mL, 112.0 mmol) slowly. The reaction mixture wasstirred at 0° C. for 30 minutes then room temperature for 4 hours. Thereaction was cooled to 0° C. and 250 mL of methanol was added slowly.The mixture was stirred at 0°0 C. for 30 minutes and excess solvent wasremoved under vacuum. The resulting oil was chromatographed by 15% ethylacetate in hexanes on silica gel to give 10.59 g (84%) of the alcohol(6) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ7.37 (m, 5H), 5.14 (s, 2H) 3.57 (ABX, 2H,J_(AB)=10.99 Hz, J_(AX)=4.34 Hz, J_(BX)=6.85 Hz, ν_(AB)=51.71 Hz), 2.42(ABX, 2H, J_(AB)=15.26 Hz, J_(AX)=7.60 Hz, J_(BX)=5.56 Hz, ν_(AB)=18.81Hz), 2.15 (m, 1H), 1.87 (br s, 1H), 1.63 (m, 1H), 0.93 (m, 2H), 0.88 (d,3H, J=6.15 Hz), 0.87 (d, 3H, J=6.45 Hz).

Step 6

To a solution of the alcohol (6) (10.22 g, 40.82 mmol) in 50 mL ofanhydrous pyridine at 0° C. was added tosyl chloride (8.60 g, 45.11mmol). The reaction mixture was stirred at 0° C. for 15 minutes thenstood overnight in a refrigerator at 4° C. The reaction mixture wasdiluted with 160 mL of ethyl acetate and 100 mL of water. The mixturewas cooled to 0° C. in an ice-water bath and then concentratedhydrochloric acid was added slowly to neutralize excess pyridine (untilpH 2). The organic layer was collected and the aqueous layer wasextracted with ethyl acetate (3×100 mL). The combined organics weredried with anhydrous magnesium sulfate and then filtered. The resultingpale yellow oil after concentration was chromatographed by 10% ethylacetate in hexanes on silica gel to give 14.44 g (87%) of the tosylate(7) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ7.77 (d, 2H, J=8.27 Hz), 7.34 (m, 7H), 5.07(s, 2H), 4.00 (ABX, 2H, J_(AB)=9.77 Hz, J_(AX)=4.07 Hz, J_(BX)=65.9 Hz,ν_(AB)=27.58 Hz), 2.44 (s, 1H), 2.44-2.20 (m, 3H), 1.46 (m, 1H),1.28-1.02 (m, 2H), 0.81 (d, 6H, J=6.58 Hz).

The tosylate (7) was also prepared from acid (5) in an alternate method.This method was advantageous over the previous procedure above since itminimized the amount of β-isobutyl-γ-lactone produced as a by-product inthe reaction above.

A solution of acid (5) (22.3 kg, 84.4 mol) in methyl-t-butyl ether (198kg) was cooled to −6° C. Borane-methyl sulfide complex (15.6 kg, 177mol) was added while maintaining a reaction temperature of 5° C. orless. The mixture was then warmed to 20° C. and stirred for two hours.The mixture was cooled to 0° C. and methanol (24 L) was added whilemaintaining a reaction temperature of 5° C. or less. Water (132 L) wasadded at a temperature of 15° C. or less. The phases were separated andthe aqueous phase was extracted with methyl-t-butyl ether (27 kg). Theorganic were combined and extracted with water (72 L). The solution wasconcentrated to an oil by distillation and ethyl acetate (23 kg) wasadded. The solution was again concentrated to an oil by distillation togive alcohol (6). Pyridine (53 kg) was added. The solution was cooled to1° C. and para-toluenesulfonyl chloride (23 kg, 121 mol) was added whilemaintaining a reaction temperature of −5° C. to 5° C. The mixture wasstirred at 2° C. for 8 hours then warmed to 20° C. Water (12 L) wasadded while maintaining a reaction temperature of 23° C. or less. Themixture was cooled to 1° C. and aqueous hydrochloric acid (52 kgconcentrated acid in 63 L water) was added. Methyl-t-butyl ether (296kg) was added and the mixture was warmed to 18° C. The phases wereseparated and the aqueous phase was extracted with methyl-t-butyl ether(74 kg). The organic phases were combined and extracted with aqueoushydrochloric acid (0.6 kg concentrated hydrochloric acid in 20 literswater), aqueous sodium bicarbonate (2.7 kg sodium bicarbonate in 50liters water), and water (30 L). The organic solution was concentratedto an oil by distillation. Methyl-t-butyl ether (19 kg) was added andthe mixture was again concentrated to an oil. The resulting product wasdissolved in methyl-t-butyl ether (37.9 kg) and stored as a solution.Weight of tosylate (7) contained in methyl-t-butyl ether solution 30.1kg (88% yield).

Step 7

A mixture of the tosylate (7) (14.44 g, 35.70 mmol) and sodium azide(5.50 g, 84.59 mmol) in 180 mL of anhydrous dimethylsulfoxide was heatedat 65° C. overnight. The reaction mixture was cooled to room temperatureand 900 mL of water was added. The mixture was extracted (4×) with atotal of 2 L of hexanes. The combined organic extracts were dried withanhydrous magnesium sulfate and then filtered. The filtrate wasconcentrated and the resulting oil was then chromatographed by 8% ethylacetate in hexanes on silica gel to give 8.55 g (87%) of the azide (8)as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ7.37 (m, 5H) 5.14 (s, 2H), 3.33 (ABX, 2H,J_(AB)=12.27 Hz, J_(AX)=4.95 Hz, J_(BX)=6.10 Hz, ν_(AB)=22.87 Hz), 2.39(m, 2H), 2.19 (m, 1H), 1.62 (m 1H), 1.20 (m, 2H), 0.88 (d, 6H, J=6.44Hz).

Step 8

To a solution of the azide (8) (8.55 g, 31.05 mmol) in 500 mL oftetrahydrofuran was added 62 mL of a 1N aqueous hydrochloric acidsolution and 1 g of 10% palladium on carbon catalyst. The mixture wasshaken overnight at room temperature on a Parr apparatus. The catalystwas removed by filtration over a pad of celite. The filtrate wasconcentrated and 50 mL of a 1N aqueous hydrochloric acid solution wasadded. The aqueous solution was wash ed with ether (3×50 mL). Theaqueous layer was collected and then chromatographed on a Dowex 50W×8(H⁺ form) column eluted with a 0.5N ammonium hydroxide solution.Fractions containing the amino acid (Ninhydrin positive) were collectedand then lyophilized to give 3.2 g (65%) of the amino acid (9) as awhite solid. mp=175-176° C.; [α]D²³=10.520 (1.06, H₂O).

EXAMPLE 2 (S)-(+)-4-amino-3-(2-methyloropyl)butanoic acid

This compound was prepared in the same manner as Example 1, except thatamino acid (9) is prepared from azide (8) by a 2 step process utilizingan intermediate azide (8a) which is subsequently reduced (the 1 stepreduction approach identified as step 8 is described above). Thesynthetic procedure of Example 2 is depicted in Chart Ia.

Step 1: Preparation of Intermediate Azide (8a)

Azide (8) (10.7 g, 0.040 mol) in ethanol (100 mL) and water (20 mL) wastreated with 50% aqueous sodium hydroxide (9.8 g). The mixture wasstirred at 30° C. for 45 minutes. Ethanol was removed under reducedpressure until 30 g of liquid remained, water (100 mL) was added and themixture was extracted with methyl-t-butyl ether (4×100 mL). Themethyl-t-butyl ether extracts were extracted with 1 M sodium hydroxideand the aqueous phases were combined and acidified to pH 1.6 withconcentrated hydrochloric acid. The aqueous mixture was then extractedwith methyl-t-butyl ether (2×100 mL) and the organic extracts werecombined and concentrated under reduced pressure. The resulting oil wasdissolved in heptane (50 mL) and extracted with saturated aqueous sodiumbicarbonate (2×40 mL). The aqueous extracts were extracted with heptane(50 mL), combined and acidified to pH 1.6 with concentrated hydrochloricacid. The aqueous mixture was extracted with heptane (2×50 mL). Theheptane extracts were extracted with water (40 mL), combined, andconcentrated under reduced pressure to give 5.4 g (75%) of intermediateazide (8a) as an oil.

¹H NMR (200 MHz, CDCl₃): δ10.8 (br s, 1H), 3.36 (m, 2H), 2.38 (m, 2H),2.18 (m, 1H), 1.64 (m, 1H), 1.25 (m, 2H) , 0.91 (d, 6H, J=6.56 Hz)

Step 2: Synthesis of Amino Acid (9) from Intermediate Azide (8a)

Intermediate azide (8a) (12.7 g, 68.6 mol) was dissolved inmethyl-t-butyl ether (80 kg). The mixture was subjected to catalytichydrogenation in the presence of 5% palladium on carbon (2.0 kg of 50%water wet) at 49 to 55 psi of hydrogen until intermediate azide (8a) hasbeen consumed. The mixture was filtered and the solid was washed withmethyl-t-butyl ether (30 kg). The solid was dissolved in a solution ofhot isopropanol (75 kg) and water (60 kg) and the solution was filtered.The isopropanol water solution was cooled to −3° C. and the product wasfiltered and washed with cold isopropanol (16 kg). The solid was driedunder reduced pressure to give 6.4 kg (59%) of amino acid (9).

This reduction may be conducted in a variety of solvents. Successfulreductions have been carried out in heptane, ethanol/water, isopropanol,isopropanol/water, methanol/water, and tetrahydrofuran/water as well asmethyl-t-butyl ether.

EXAMPLE 3 (S)-(+)-4-amino-3-(2-methylpropyl)butanoic acid

The following “steps”, refer to Chart II. All reactions were carried outunder an atmosphere of nitrogen.

Step 1

To a solution of 4-methylvaleric acid (50.0 g, 0.43 mol) in 100 mL ofanhydrous chloroform was added thionyl chloride (60 mL, 0.82 mol). Thereaction mixture was refluxed for 2 hours and then cooled to roomtemperature. Excess chloroform and thionyl chloride was removed bydistillation. The residue oil was then fractionally distilled to give45.3 g (78%) of the acid chloride (102), bp=143-144° C.

Acid chloride (102) was also prepared by an alternative method whicheliminated use of chloroform which has waste disposal and operatorexposure difficulties. The alternate method also minimized the formationof 4-methylvaleric anhydride.

To a solution of thionyl chloride (98.5 kg, 828 mol) andN,N-dimethylformamide (2 kg, 27 mol) was added 4-methylvaleric acid (74kg, 637 mol) while maintaining a reaction temperature of 25-30° C.Hexanes (30 L) were added and the solution was maintained at 30° C. to35° C. for 1 hour and 15 minutes. The solution was then heated to 70° C.to 75° C. for 1 hour and 10 minutes. The solution was subjected toatmospheric distillation until a solution temperature of 95° C. wasreached. After cooling, hexanes (30 L) were added and the solution wassubjected to atmospheric distillation until a solution temperature of97° C. was reached. Distillation of the residual oil produced 79 kg(92%) of acid chloride (102), bp=77° C., 60-65 mm Hg.

Step 2

To a solution of (4R,5S)-(+)-4-methyl-5-phenyl-2-oxazolidinone (5.27 g,29.74 mmol) in 70 mL of anhydrous tetrahydrofuran at −78° C. under argonatmosphere was added a 1.6 M solution of n-butyllithium (19 mL, 30.40mmol) in hexanes slowly. The mixture was allowed to stir at −78° C. for15 minutes then the acid chloride (4.5 g, 33.43 mmol) was added toquench the reaction. The reaction was stirred at −78° C. for 10 minutesthen 0° C. for 30 minutes. A saturated solution of sodium bicarbonate(50 mL) was added and the mixture was stirred at 0° C. for 30 minutes.The organic layer was collected and the aqueous layer was extracted withethyl acetate (3×). The organic extracts were combined and dried withanhydrous magnesium sulfate. It was then filtered and concentrated togive a colorless oil. The oil was then chromatographed with 8% ethylacetate in hexanes on silica gel to give 7.56 g (82%) of theacyloxazolidinone (103) as a white solid. Anal. Calcd for C₁₆H₂₁NO₃: C,69.79; H, 7.69; N, 5.09. Found: C, 69.56; H, 7.63; N, 5.06.

Acyloxazolidinone (103) was also prepared by an alternate method whichwas conducted at −5° C. to 0° C. rather than −78° C. which is difficultand expensive to achieve on a manufacturing scale. The alternate methodalso gave a crystalline solid from the reaction mixture a rather than anoil which must be chromatographed.

To a solution of 4-methyl-5-phenyl-2-oxazolidinone (64 g, 0.36 mol) inanhydrous tetrahydrofuran (270 g) at −5° C. was added a 15% solution ofn-butyllithium in hexane (160 g, 0.37 mol) over a temperature range of−5° C. to 0° C. Acid chloride (102) (48.6 g, 0.36 moi) was added at −10°C. to 0° C. The reaction was quenched with a solution of water (90 mL)and sodium bicarbonate (4 g). Ethyl acetate (200 g) was added and thelayers were separated. The organic layer was extracted with water (2×50mL) and the aqueous phases were back extracted with ethyl acetate (100g). The organic extracts were combined and approximately 150 mL ofsolvent was removed by distillation. Atmospheric distillation wascontinued and heptane (2×200 g) was added until a vapor temperature of95° C. was reached. The solution was cooled to 5° C. The product wascollected by filtration, washed with cold heptane, and dried to give 79g (80%) of acyloxazolidinone (103).

Step 3

To a solution of diisopropylamine (7.6 g, 0.075 mol) in anhydroustetrahydrofuran (10 mL) at 0° C. under nitrogen was added a 1.6 Mn-butyllithium in hexane (47 mL, 0.075 mol) while maintaining atemperature of −5° C. to 0° C. The resulting solution was added to asolution of acyloxazolidinone (103) (18.6 g, 0.068 mol) intetrahydrofuran (160 mL) at −55° C. to −45° C. The solution was stirredat −55° C. to −45° C. for 30 minutes. The solution was then added to asolution of t-butyl bromoacetate (14.6 g, 0.075 mol) in tetrahydrofuranat −55° C. to −45° C. The solution was cooled to −65° C. and allowed towarm to 10° C. over a period of 2 hours. The reaction mixture wasquenched with the addition of saturated aqueous ammonium chloride andextracted with ethyl acetate. The organic layer was dried (MgSO₄),filtered, and the solvent was removed under reduced pressure. Theresidue was recrystallized from heptanes, filtered, and dried underreduced pressure to give 18 g (68%) of acyloxazolidinone (104).

¹H NMR (200 MHz, CDCl₃): δ7.4-7.2 (m, 5H), 5.65 (d, 1H, J=7.09 Hz), 4.74(m, 1H), 4.26 (m, 1H), 2.69 (m, 1H), 2.44 (m, 1H), 1.65-1.45 (m, 2H),1.39 (s, 9H), 0.93 (m, 6H), 0.89 (d, 3H, J=7.87 Hz).

Alternatively, the order of addition of the reagents may be reversed.t-Butyl bromoacetate may be added to the solution containingdiisopropylamine, n-butyllithium and acyloxazolidinone (103). The finalproduct isolation may also be conducted by doing a distillation andreplacing the solvents present (hexane and tetrahydrofuran) withisopropyl alcohol. Acyloxazolidinone (104) then crystallizes from theisopropyl alcohol solution. The following experimental procedureillustrates this alternative.

To a solution of diisopropylamine (23.1 g, 0.229 mol) in anhydroustetrahydrofuran (30 mL) at 0° C. under nitrogen was added a 2.5 Mn-butyllithium in hexane (92 mL, 0.229 mol) while maintaining atemperature of −5° C. to 0° C. The resulting solution was added to asolution of acyloxazolidinone (103) (60.0 g, 0.218 mol) intetrahydrofuran (400 mL) at −45° C. to 40° C. The solution was stirredat −45° C. to −40° C. for 30 minutes. t-Butyl bromoacetate (44.6 g,0.229 mol) was then added to the reaction solution at −45° C. to −40° C.The solution was allowed to warm to 10° C. over a period of 2 to 3hours. The reaction mixture was quenched with the addition of saturatedaqueous ammonium chloride. The organic layer was separated from thewater layer. The solvent was removed under reduced pressure and replacedwith isopropyl alcohol. The product crystallized from isopropyl alcoholand was filtered and dried under reduced pressure to give 53.8 g (63%)of acyloxazolidinone (104).

Step 4

To a precooled (5° C.) solution of acyloxazolidinone (104) (60.0 g, 0.15mol) in tetrahydrofuran (266 g) was added a solution of 30% hydrogenperoxide (71 g), 9.4 g lithium hydroxide monohydrate (0.22 mol) andwater (120 mL) over a period of 35 minutes so as to maintain a reactiontemperature of 5° C. The mixture was stirred at 3-5° C. for 2.5 hours.The reaction was quenched by addition of a solution of sodium sulfite(50 g), sodium bisulfite (27 g), and water (310 mL) at a temperature ofless than 29° C. Reptane (100 mL) and methyl-t-butyl ether (100 mL) wereadded and the layers were separated. The aqueous layer was extractedwith methyl-t-butyl ether (100 mL) and the organic layers were combined.The solvent was replaced with heptane by distillation and the resultingheptane solution (400 mL) was cooled to 5° C. The resulting solids werefiltered and the filtrate was extracted with warm water (2×150 mL, 1×200mL, 1×300 mL). The solution was concentrated by evaporation to give 34.5g (97%) acid (105) as an oil.

¹H NMR (200 MHz, CDCl₃): δ11.5 (br, s, 1H), 2.85 (m, 1H), 2.67-2.29 (m,2H), 1.60 (m, 1H), 1.44 (s, 9H), 1.32 (m, 2H), 0.92 (m, 6H).

Step 5

Acid (105) (72.4 g, 0.314 mol) was dissolved in tetrahydrofuran (360 mL)and cooled to 0° C. A 2.0 M solution of borane dimethylsulfide complexin tetrahydrofuran (178 mL, 0.356 mol) was added at 0° C. The solutionwas allowed to warm to 48° C. then cooled to 25° C. After 2 hours and 45minutes, the reaction was quenched with the addition of methanol (300ml) and the Isolvent was removed under reduced pressure. Additionalmethanol (300 mL) was added and the solution was concentrated underreduced pressure to give 66 g (97%) of alcohol (106) as an oil.

¹H NMR (500 MHz, CDCl₃): δ3.62 (m, 1H), 3.45 (m, 1H), 2.44 (br s, 1R),2.36-2.21 (m, 2H), 2.05 (m, 1H), 1.64 (m, 1H), 1.45 (s, 9H), 1.24-1.04(m, 2H), 0.91 (m, 6H).

Step 6

Alcohol (107) (51.9 g, 0.24 mol) was dissolved in pyridine (130 mL) andcooled to 5° C. p-Toluene sulfonyl chloride (57.2 g, 0.30 mol) was addedand the mixture was stirred at 22° C. for 21 hours. The reaction wasquenched with the addition of water (95 mL) and 18% aqueous hydrochloricacid (300 mL) at less than 300° C. Methyl-t-butyl ether (350 mL) wasadded and the layers were separated. The aqueous layer was extractedwith methyl-t-butyl ether (350 mL). The organic layers were combined,washed with 1% aqueous hydrochloric acid (2×100 mL), saturated aqueoussodium bicarbonate (1×150 mL), and water (1×100 mL). The organicsolution was treated with decolorizing charcoal, filtered, andevaporated to give 77 g (86%) of the tosylate (107) as an oil.

¹H NMR (200 MHz, CDCl₃): δ7.78 (d, 2H, J=8.25 Hz), 7.34 (d, 2H, J=8.25Hz), 3,96 (m, 2H), 2.45 (s, 3H), 2.32-2.12 (m, 3H), 1.6-1.4 (m, 1H),1.40 (s, 9H), 1.2-1.1 (m, 2H), 0.83 (m, 6H).

Step 7

Tosylate (107) (65 g, 0.175 mol) was dissolved in dimethyl sulfoxide (40mL). The dimethyl sulfoxide solution along with additional dimethylsulfoxide (10 mL) was than added to a solution of sodium azide (11 g,0.26 mol) in dimethyl sulfoxide (450 g) at 63° C. The mixture was thenstirred at 65° C. for 6 hours. Water (140 mL) and heptane (250 mL) wereadded to the reaction and the layers were separated. The aqueous layerwas extracted with heptane (250 mL) and the organic layers werecombined. The solvent was removed under reduced pressure to give 42 g(95%) of the azide (108) as an oil.

¹H NMR (200 MHz, CDCl₃): δ3.32 (m, 2H), 2.22 (m, 2H), 2.15 (m, 1H), 1.63(m, 1H), 1.46 (s, 9H), 1.19 (m, 2H), 0.89 (m, 6H).

Step 8

Azide (108) (36.3 g, 0.15 mol) was placed in 88% aqueous formic acid(365 mL). The mixture was stirred at 30° C. for 4.5 hours. Decolorizingcharcoal was added and the mixture was filtered and concentrated underreduced pressure to give an oil. Heptane (250 mL) was added and themixture was vacuum distilled to give an oil. Water (125 mL) and heptane(250 mL) were added and mixed vigorously. The layers were separated andthe water layer was washed with heptane (250 mL). The heptane layerswere combined and concentrated under reduced pressure to give 24.6 g(88%) of intermediate azide (108a) as an oil.

Alternatively, aqueous hydrochloric acid may be used rather than aqueousformic acid in order to conduct the hydrolysis.

Step 9

Intermediate azide (108a) (12.7 g, 68.6 mol) was dissolved inmethyl-t-butyl ether (80 kg). The mixture was subjected to catalytichydrogenation in the presence of 5% palladium on carbon (2.0 kg of 50%water wet) at 49-55 psi of hydrogen until intermediate azide (108a) hasbeen consumed. The mixture was filtered and the solid was washed withmethyl-t-butyl ether (30 kg). The solid was dissolved in a solution ofhot isopropanol (75 kg) and water (60 kg) and the solution was filtered.The isopropanol water solution was cooled to −3° C. and the product wasfiltered and washed with cold isopropanol (16 kg). The solid was driedunder reduced pressure to give 6.4 kg (59%) of amino acid (109).

This reduction may be conducted in a variety of solvents. Successfulreductions have been carried out in heptane, ethanol/water, isopropanol,isopropanol/water, methanol/water, and tetrahydrofuran/water as well asmethyl-t-butyl ether.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously many modifications and variations of the present invention arepossible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims whereinreference numerals are merely for convenience and are not to be in anyway limiting, the invention may be practiced otherwise than asspecifically described.

What is claimed is:
 1. A method of treating a patient having Parkinson'sDisease which includes administering to a patient an anti-seizureeffective amount of the compound 4-amino-3-(2-methylpropyl) butanolcacid.
 2. A method of treating a patient having Parkinson's Disease whichincludes administering to a patient an anti-seizure effective amount ofthe compound S-(+)-4-amino-3-(2-methylpropyl)butanoic acid.